This application is part of an overall effort of our laboratory, which seeks to elucidate the control of neuroendocrine differentiation in normal thyroid C-cells and their neoplastic counterpart, medullary thyroid carcinoma (MTC). Understanding this control will provide a grasp of what functions may be altered in tumor developement and progression. Medullary thyroid carcinoma is a good model for this study, since defined stages of progression can be identified. We have defined an in vitro system to study these steps of MTC differentiation and progression. MTC cells in culture can be differentiated with respect to many characteristics of normal C- cells, either chemically or by ras oncogene introduction. Differentiation in turn can be blocked by myc oncogene introduction. The current Proposal will further characterize these findings, by cell biology, biochemistry, and molecular biology, concentrating on mechanisms of differentiation. 1. The effects of normal and activated cellular rasH oncogenes on MTC cell differentiation will be investigated, to determine whether the effect is specific to viral rasH. The effects of rasH on CT gene transcription will be ascertained. The effects of the myc oncogen on TT cells, and on ras induced differentiation, will be characterized. Finally, the effects of blocking endogenous c- rasH and c-myc expression will be investigated, using antisense RNA. 2. The biochemical pathway of ras-induced differentiation and myc blocking of differentiation in MTC cells will be studied, by assaying several enzymes and messengers in signal transduction pathways. Identifying alterations in these pathways in differentiated cells, or in cells blocked in differentiation, will be important in defining the processses controlling neuroendocrine differentiation. 3. The DNA sequences controlling ras mediated induction of CT gene expression will be defined, and compared to sequences involved in TPA or cAMP induced CT gene expression, using gene transfer and DNA binding assays. These same assays, along with DNA affinity chromatography, will then be used to partially purify a relevant DNA binding protein involved in ras mediated CT gene induction. This study will be important in elucidating the mechanisms of ras function in cells.